IMMORTALIZED HUMAN HEPATOCYTES AS A TOOL FOR THE STUDY OF HEPATOCYTIC(DE-)DIFFERENTIATION

Primary human hepatocytes were immortalized by stable transfection wit h a recombinant plasmid containing the early region of simian virus (S V) 40. The cells were cultured in serum-free, hormonally defined mediu m during the immortalization procedure. Foci of dividing cells were se en after 3 months. Albumin- and fibrinogen-secreting cells were select ed and cloned by limiting dilution to obtain homologous cell populatio ns. The established IHH (immortalized human hepatocyte) cell lines wer e evaluated for their usefulness in studying the regulation of cell gr owth and of certain differentiated hepatocyte functions. IHH cells ret ain several differentiated features of normal hepatocytes. They displa y albumin secretion at a level comparable to cultured primary human he patocytes (30 mu g albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous org anic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells f or the study of localization of the newly characterized multidrug resi stance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differ entiated feature of short-term cultured primary hepatocytes which is n ot detectable in IHH cells is active uptake of the bile salt taurochol ate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoprotein s, apolipoprotein B (0.6 mu g/ml per day), and apolipoprotein A-I (1 m u g/ml per day). However, they secrete apoB-containing TG-rich lipopro teins mainly in the LDL density range, while short-term cultured prima ry hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range. In conclusion, functions that are rapidly lost in short-term h epatocyte cultures are, in general, not displayed by IHH cells. Immort alized human hepatocytes provide a valuable tool for studying the regu lation of hepatocyte proliferation-related phenomena.